Purification of potato virus X white clover mosaic virus, tobacco mosaic virus and ribosomes by column chromatography

.I..

.

JOllYi~~ll Of Clll~l~~~7~ll~~~~~~~l~~ 75 (1973) 235-244 0 Elscvicr Scicntilic I?ublishin~ Compnny, Amstcrdarn-

Printed

in ‘I’hc Nctlicrlnntla

CHIIO?iI. 0373 I?UR1I~ICATION OF POTATO VIRUS X, WHITE CLOVER MOSAIC VIRUS, TOBACCO MOSAIC VIRUS AND RIBOSOMES BY COLUMN CI-IROhIATOGRAPHY

Clironiatogral~l~ic proccdurcs for tlic isolation on cellulose columns of potato *virus S, tobacco mosaic virus and ribosomcs from tobacco plants, and of white clover mosaic virus ~1x1 rhsomcs from bean plants, are described. The yields obtained by using proccdurcs involving the use of ten different solvents were compared, Tlic first step consistecl of the extraction of homogenates, each preparecl from x00 g of fresh plant matcrinl, by using columns paclrecl with 20 g of grouncl cliromatographic paper ancl clution with ten cliffcrent solvent systems. Tlic compositions of these solvents differccl in their contents of clextran, sucrose, tris(llydroxymethyl)amit~omcthanc-acetic acid buffer (pH 7). magnesium chloride, magnesium acetate, soclium clictl~ylclitlliocnrbamate, urcn and polyethylene glycol. The seconcl step consistecl of fractionation of viral nncl ribosomal nucleoproteins ancl otlicr UV-absorbing substances in the effluents from the first columns. Elution was cnrriccl out on coluks containing IO g of Whatman cellulose powder in which a stepwise reduction in polyetllylcnc glycol, sodium chloride, sodium acetate, nmmonium ncetatc and magnesium acetate was appliccl, This proceclure rcsultccl in the complete separation of virus particles and ribosomes. Using the UV absorption of tlic effluents ns a measure. the most effective procedure for the recovery of viruses and ribosomcs was founcl. The extraction solvent in the first step contained IO/” clestran, 0.4 M sucrose, 0,0x AI tris(liyclroxymetl~yl)aniinometliane-acetic acid buffer of p1-I 7 and 0.1% sodium clietl~vlditl~iocarbamate. In the second step, tllc omission of magnesium acetate resulted in the libcrntion of ribosomes.

In previous

,. ._. _. _.-. I+xcnt niicltd faltulty, l

papers,

VENEICAMP

nclclross: Vysolt;i Sucliclol II Prnhy,

AND

Moscw *W-3described

Skoln Zcm~clLZlslcn V Pmzc, Czccliowlovdtin.

Kntcclra

the

Oclirany

purification I
of

Agrono-

Js I-!. VENEICAMI’,

236

W.

H.

M.

RIOSCH,

V. TABORSIW

smic plnnt viruses by using cliromatogral~l~y on cellulose colunins vAtI solutions ctmtnining pc~lyctliylene glycol ins solvents. Ilowever, mny interfere in tlic puril’lic prcsencc of plicnolic compounds, fication of viruses. LOOMIS AXII BATTAILE~ rcportcd tlie rcvcrsiblc ndsorption of l~lwnolic compounds on proteins by llydrogen bonding. After osidation of the pllenc~ls, tllis adsorplion 1xcamc irrcversiblc 11.1 covalent condensntion. Oxidation ciui ncvertliclcss be avoitlcd by tlie clirt~ination of polyl~licnol osiclnse. Scvcrnl invcstic~ietl~y1clitliiogntors lmriticd plant viruses witli niore success 13)7 using sodium ciubuiintc to cliniinntc this cnzymc ; for csainplc: prune dwarf md sour cllerry riccrotic ringspot viruses wcrc purilietl by Hnnrmox Am I.%LToNB*U, Tularc npple cucunibcr rnosnic virus by I-ImnIsoN ANI> inosaic virus by MINI< Ml3 Bmmo1+, lJIlsIm21xPJJ, cocm swollen-shoot virus by BI~UNT AND I
RIISTI10%

Eiglit-weeks-old “Wllite Burley” tobacco plants were n~eclxmically inoculated wit11 potato virus S am1 witli a niisture of potato virus S nncl tolmzco mosaic virus. Sk-\vcel
PURIFICA’I’ION

01: VIRUSJES ANJ) RIJ3OSOMJZS RY cc

‘237

mosaic virus, Ten clays after inoculation, x00 g of frcsli leaves were taltcn for catch procedure described below. In tlwce exIxrinients, Jlinc diffcrcnt, clJrornn.togmplJic proccclurcs were compared with the original proccclure of VJZNJSKABJP ANJ) Moscw~, which is rcfcrrcd to as procedure 0. For en1211csIwiJncnt, fresIJ plant Jnatcrial was harvcstccl, cut into small pieces of about 1 cnJ2, rancloinizcd ancl tlwn cliviclccl into ten portions, cacli containing I00 g of plant mutcrial. One portion WEB used for proccclurc o. AnotlJcr portion was homogcJiizcc1 in a misture of I00 ml of double-concentratecl and 150 1111of singlc-conccntrated solvent, the composition of which is inclicatccl in Table I uncler proccclure I. lhcli of the other portions wns lioJt~ogcnizccl with tlic solvent of tlic corresponding proccclure as in Table I. Then each liomogcnatc wns filtcrecl through nylon cloth (Itlcmoclur nortnal 500, Vcrsciclag, Krcfelcl, G.F.R.) nncl tlic filtrate was usccl.

First

columns:

Iioight.

N;I.-J>lX

20 g of ground

DWtlXll

=

~Vll;~tm;rn No. I chrorllatogral~llic pnpcr, 5-cm cliiblllctcr, IO-cm n&I bulTcr; Tris = tris(l~vtlrosymcthyl)anlinornctJ~~~n~-~~~tic glycul fjooo. tlicltlplclitllioc~~r~~~~~~~~t~; J)Ih = polycttlylctw _....... .__

tlcxtrall

.= sotlium

Cllc,,ricf/l

. Jhxtmn Sucrose ‘L’ris, 1’1-17 MgCI, My ncctlltl? ;;ib~J~c

(‘j/o) (M) (AI) (M) (M) I;%;

J)JS;; (SP) _......__..._.. _ .

T_-jOO:

PvocPo?llm . _ _ ^_ .. . r 3

_.._..

I O#.l 0.01 0.004

. .. .

_

_.

.

3

.

._.._

4 ._

_

5

T 00‘1 oeor

x 04 cJ,v1

I

I

O.‘l

O,.#

O.OI

0,or

0.004

0,004

0.00.~ 0.1

0.

r

1 .

.-._-..._. ..- _._

. __-_.... .

..__-._.

. . . _,_

.,,“_._

0.1 I 0*5 ..,.. ..- _...

One sliect of C\llx-WiiaJ~ No. 1 cliromatojirapliic paper was cut itito picccs of shout 10 cm2 and ground in 500 nil of water with rtn electric stirrer (I-IcicloIl~1~ clcctro kg, Kelheim, laboratory stirrer 1’ypc E%I;Z). The slurry was poured irlto tubes of 5-cm cliamctcr containing a l)lug of cotton-wool at the bottom, The height of the settlccl columns was 10 cm. Eacli column was waslied with 400 ml of 0.1% sodium ctlJylenecliaminctetruacetatc solution followed ljy 400 ml of water. IYnally, tlw columns were ecluilil~ratccl with 200 nil of the solvent clescribccl iii Table I. The filtrates from the homogcnatcs were nppliecl on to the corrcspancling columns. During the clutions, the first ISO in1 of the cthent were cliscarclecl. Tllc clution was coiltinuccl with the corrcsponc1iJi.g solvent (Tz~blc I) until tlic volume of the effluent was go0 ml. Polyetllylcne glycol w;1s rtdclcd to each effluent up to I\. fm1.1 concentration of 5 y,, To the effluent obtairlecl by usitlg the first proccclure, soclium chloride was adclecl to give a fin:ll concentration of zo/,. Sodium acetntc was added to the eilluent by applying t1Jc second procedure up to a finill concentratiotl of z”/“* To the effluent ncetatc WL~S ndclecl up to il. fiual obtained by using the third Iwoccclurc, ammonium

J, II. VISNEI~AMP, W. H. &I. MOSCH, V. TABOIZSKY

23s

firststeps

l’lic

ClL’Strilll Tlic

‘~joo,

sccrmcl

ill72

step

iii the cllluctits sccollLl

colllllllls:

clirorii~~toyr~rpllic I’ISC

=

=

cstractiotis

OS.+ n’f

of

IlOlllOJQ2lliltL%,

ilIlt

the

CilCll

SUbStallIX!S

of giVlY1

100

g of

ill

Iid1

‘.L’:lbll!

plant

llli~tcri:ll.

by

solVcllts

containing

t’%,

1.

is il I’roctionntiun of viral uticl ril.~oxoriinl riuclcoprotciiis nntl otlicr UY-nhorbing substirrlccs froiii llic lirst columns, 10 g of IV1 id ’ t ninn Cl? I I collulosc powlcr bctwucri r-cm IilyWS of ground Wlintmnn No, I pilpcr,

polyctliylcnc

nwgncsiuin

SIICrOSO

illXtiltU:

p3ll

glju~l Nit-IITJC

diillnctcr, 6000

: =

lo-cnl NilO;\C

socliuiii

Iiciglit, =

sodium

LlcctiLtc;

l1ictl~ylclitl~iocirrl~~l1llnto;

NI-I.SO:\c

I

Ywrig

=

i~1n1iin1iiun~

LKZUtiltc;

Mg(C)Ac)2

tris(liyclros~l1Ictl1~l);rlninon1ctlii~1lc

0.0

1

0.01 0.01 0.0

I

0.01 0.01 0.01

‘2

0.01

I

0.01 0.01 0.01 0.01 0.01 0.01

2

0.01

I

o.p1 0.01 0.01 0.01 0.01 0.01

4

0.01

I

0,Ol 0.01 0.01 0,or 0,Ol

5

I

0.1

I

0.01 0.01 0.01 0,OI 0301 0.01 0,Ol

0. I

0001 0001 0,OI 0,OI 0,OI 0,Ol 0801 0,OI

PURIFICATION

OF VIRUSllS

AND

RIBOSOMISS

BY

cc

‘,

239

concentration of 2%. Ammonium acetate was aclclccl’to cnch of the other cflluenCs to a final concentration of 1 %a Also, magnesium cuxtatc ~8s aclclccl to tlic cfl-luents obtained by using proccclurcs 7, 8 and g to a. ftnal concentx~tion of 0.004 M. A sccoi~cl set of columns was prcparecl by grinding up clironiatogrepliic paper ~1s clcscribccl earlier. Ten milliliters of tllis slurry were pourecl into a tube of 3 cm clinmcter wit11 a plug of cotton-wool at tlic 1~otlom. 13sccss water was removed and u suspension of 10 g of Whatman Cl; Ix ccllulosc powder in I00 ml of water wks pourccl into the tube. Aftxr allowing the tubes to stand for I5 min, part of the csccss wntcr was pcrcolatccl, allowing tlic cellulose to Ixcome compacted, A further I0 ml of grouncl cliroiiiatogra],hic paper were aclclccl, Aftxr coniplctc percolation of the excess water, tlic columns were wnsliccl witll I00 ml of 0.1% sodium elliylcnecliun~inetetraacctatc solution and then with x00 ml of water. TIE columns were equililxx.tecl with 50 nil of tlie corrcslxmcliiig first sulveiits (Table 11). T11c cfflucnts from tlic first columns were then tlppliccl on to the second columns, as clescrilxcl above. After percolation of the effluents, the sccoiicl columns were washecl with 200 ml of cc~rresponcliiig sdvcnts inclicatccl by the iiunil1er I in Table II, Finally, zoo-nil portions of solvents listccl in Tal~le II were passed successively tlwough the colunins, The UV absorbance of each eflluent was measured in a Beckman DB-G spectropl~otomctcr with ;i Sargciit recorcler. ISacli of the corrcsponcliiig solvenls was used as reference. Table III &ves tile following cliaractcristics of the spectra of viruses ancl ribosomcs: wnvelengtlls of masinium ancl minimum nbsorlxmces, the ratios of masimum and minimum absorbanccs, ancl the ratios of the alxorbances at 280 nm and tha masimum absorlxmccs.

Cllr\RACl’l~RlS’I’ICS TIIIE

T\VO-S’PRI’

01’

Tl-II3

uv

,\lGlOlzI”rIc~s

CllROillA’1’O0RA1’1~lIC

SPlfC’PRA

I’ROClSlJURlSS

OS

01’

Tl-II3

vIRuslss

C11LLUl.OSTS

r\Sl>

RIIiOsfJ~Il~s

PURll~IIEr~

13’i

COl.ULlSS

‘lb results arc avcrngcs from tlio mca4iwc) given bg ITip, I-J. nrcnwntn on the virus- i~ntl ril,osolnr!-carr~iii~ clflucnt?r. -.__..--._.__. I_... . _...-. - __.._...__._..-..._____..--~-.“.~__-.-..-___.__.-____..- _.-_ .. .._.- -.. -__-_..-.--.----.-------fr’ctfio 01 Niwfeopmlci~r IVtrvrlc~qllr vf IVni~clozgt11 I-$ J?ctl.io [I/ W!JSUJ’iJtlJJCC a/ m~xiwuw JJliJfiJJJJJJJJ ~JJl~IXitJlllJJ~ (/J/d

Cl~roinntojiriq~liic procctlurcs

_._.

.._._,____.-

._._.- .

. -.. .

lJot:rto virus.X LVhitc clover mosaic virus Tobacco niomic virus Tobacco ribosonics Wcnn ribosonics --.__-._--11--11-

dJ.WJJ’~tifJJl

tJbSW’/J/iIJJJ

JJliJ,iJJJJ#,Jl

380

(It

(~11~))

cr6soAJrrJ/cL%

?Jlll.Yi~JiUlJl

_.

111)

_.__..-_.

.

.,.

2Go ?.fr, ?.+6 260 24s 2.55 2x5 2.76 2$3 z3ti .____-..~~__~-_.--_-_-_~__--.~--

_..... .-- -

11JJJ CtJld

dJSOJ&J

I.15 1.X.5

* 0.83

I .oY

0.80

I.30

0.00

I.54

JICC

_--- . . -- .._.- .____-__-_-.__..............-.. *_. o.Ya

.-- _-_-.---.

0.59 ---p-

The criteria for l&c occurrcncc of viruses iii the cf~lucnts from the scconcl colunii~s are bad on electron microscopic studies, absorption spectra ancl infectivity for susceptible host plants. The criteria for the presence of ribosomes arc limitecl to electron micrographs and absorption spectra. In the fourth esperiment, two identical filtrates were used, each prepared from zoo g of leaves from eight-weeks-olcl “White Burley” tobacco plants. The plants were inoculated with potato virus S IO clays before hnrvesting. The first filtrate was

240

J, H. VENI~KAMF,

::

W.

H. MM.MOSCH,

V. TABORSKY

1.0.

0.5 -

t.0*

0.5

I.0 0.5

1.0

0.5 I.0 0.5

L 1.0 0.5

1.0 0.5

1.0 0.5

1.0 0.5

j.0 0.5

I!\yy63$& I

. . _

,200

,500

lllO0

2100

2400

2

3

4

5

0

a700 7

3000

3.100

a

9

0 ml 5oIvenl

effluent numbaf

Fig. I, IGwztionation 0T lmluto virw S :wcl ribosorncs from “White 13urloy” tobtrcco plants by tlw second step of the two-stop chro~nntogrn~~llic lmxcclurcs on collulosc columns. The tirst StcpS arc! cstractiona uf lmmogonatcs, CilCll of 100 g of fresh plnnt mntcrinl, using columns of 20 g of ground Wlintman No. t clix~oriintoyral~liic lxqw and solvents containing I%, clcxtmn 7-500, O..I kl sucroxc and tlic subStnnccS gi\*cn in T;rblc 1. Tlw SwoncI steps iWC fractionotions of \*iral UV-ubeorbing .Substnnccs in the clllucntu from the first g of \Vll~tmiltl Cl: 1 I CCllIll0SC pO\VClW lXt\VWll I-cm paper (J-cm tlialllctc~, IO-cm Iicigllt). Compositions of tllo Solvents numbcrccl illOng tlw ;rtscixsu ilrc indicatcrl in ‘l’rd~lc I I.. Tlic yicltl of virus in tllc cllluonts iS indicirtecl a3 :I. pcrccntagc of the tnkd amount of virus in lwocctlurc 7, Tllc Sanw IloltlS for tlw yiclcl

ilIlt

riLoSOn~i~1

nuclcol~rOt.cins

witI

ColllmnS, The! suconcl columnS luycrs of ground clwomitograpliic of ribosomcs in the clllucnts. wuight of 1.48, A ribosonic 2.~2.

“White

Fractions

Rurlcy”

1,1213 Uvicortl

with

10

A virus content Content

n pluu

tobncco

other

Cfllltili!I

sign

plants.

:tLSorptiO~~tnctcr.

Of

loo~(,

contain

‘IX2

of ~oo”/~ is cquivalcnt is

cquivnlcnt

highly

~llISO~lJirllCCS

to

nn

to nn ;~bsorbnncc/ml/g

infectious virus giving Of tlic clflucnls at 25.1 nni

fresh

frcwli weight typiwl syniptonis wcrc rccordctl with

;lbSOPba1X!C/IIl!+/g

of on nn

PURIFICATION

OF VIRUSES

AND RIBOSOMIW

RY cc

241

1.0 0.0

1.0 0.0

1.0

0.0

1.0 0.6

1.0 0.6

1.0 0.6

I.9 0.5

1.c O.?

$.a 0.5

t.c 02

Pig. 2. ihctionntions of white clover Inosiric virus nnd ribosomcs from bcnn plants (“l3atllaf”) by the sccontl stop of the two-step clirouiatogrrrphic proccclurcs on ccllulosr? columns, For uxplnnittions, so0 Pig. I. lktctions with a plus sign contain higlily infcctioua virus giving typicnl symptoms on “Untanf” IJWIl phtlt% r\ virus content of x00%, ia cquiwlcnt to nn nbsorbancc/ml/g fresh WJight of 3.92. A rillcwomo content of roo(g is cquivnlcnt to nn idxtorl~nllcc/nll/g frcnh wcight of 12.@,

chromatographecl according to the above-mentioned procedure 7, and the results are shown later in the upper part of Fig. 4, TIN second filtrate w;~s chromatographed according to procedure 7 escept that the zoo-ml portions of the successive solvents with 3o/o, 2O/” and 1% of polyethylene glycol contained 0.004 M magnesium acetate. Thcsc percolations were continued by the serial passage of zoo-ml volumes of solvents

242

J. H. VISNI’:l
containing 1 OA, and oak, of plycthylcnc glycol without mngncsium acctntc arlcl finally bl* the passage of 200 ml of water. l’lic results from tllis procedure are given later in the lower part of Fig. 4. l’hc UV al~sorlxuicc of the cfflucnto was nieasurcd sl~ectrol~l~otoI~~c!trically, as dcscrilxd above.

The cf’tlucnts from the first colunins, 0, I, 2, 3, and 4. lind ;L brown colour. l’hc presence of sodium tlictliylc~itlIioc:~rl~nmatc ‘iI1 the cstroction solvent inducccl il yellO\v COlCXlr in tllC efllUCIlt. Tlic first cfflucnts from all of the scconcl colunins containccl low-molccularwciglit substances, including plicnolic conilx~unds. Tllcsc el’flUcIlts IliLd 21 very high absorption at zG0 nni. The following conclusions CiLIl lx clrawn front the results ol,~tainccl from tlIc! csperinients sliowii in I.~igs. r-3. Extraction with a solvent containing 1% of clcstran yiclclccl niucli mure lxAxt0 virus S and tobacco mosaic virus tllaii was 01Axinccl in tlic original proceclurc (nunilxr 0). This solvent, liowcvcr, was no more effcctivc in estracting white clover mosaic virus . I TlIe WC of acctatc ions, instead of chloriclc ions, in tile first solvent in the second column did not improve tile yiclcl (compare lmmdure 2 wit11 proccclure I). Wlicn tllc first solVent of the. scconcl column containecl aninionium ions insteacl of sodium ions, tlIc final result was improvccl in tlic cast of tolxuxo plants infected with potato Virus S (coniparc proccclurc 3 with proccclurc 2). The first .solvent, containing I “A, ammonium acetate, clicl not yield more virus (compare ixpccclurc 4 with proccclurc 3). Tlic prcsclicc of sodium clictliylclitlIiocurl~aniatc in tlic estrnction solvent in the first column and in the first solvent in tllc scconcl column resulted in a larger amount of virus (compare proceclurcs 5,6, 7, S and 0 with proccclurcs 0, I, 2, 3 and 4). In proccclurc 5, white clover mosaic Only tobacco mosaic virus w;~s an esception. virus was successfully clutcd with three succcssivo solvents. Omission of magnesium ions from the solvent in the second columns rcsultccl in the elutiorl of ril)osomcs (compare lxoccclurcs 6, 7, S and 9 with procedure 5). This yielded more white clover IllosiLiC virus, but not more potato virus S or tobncco mosaic Virus. I More important is the clution of potato virus S in the espcriment sliown in Fig. 1 in one fraction, and the clution of white clover mosaic virus in two fractions, When the tobacco plants were infected with tobacco mosaic virus and potato virus S, both viruses were elutecl wit31 two succcssivc solvents (compare proccclure G with procedure 5), In procedure 5 of the experiment sl~own in Fig. 2, a small amount of ribosomes was elutecl with water. Omission of magnesium ions from tlic estraction solvent ip tlic first colunin yielded more ribosomes (compare proceclurc 7 with proceclurc G) and an even larger amount of potato virus S and white clover mosaic virus. Beau p!ants containccl 5-G times as many ribosonies as tobacco plants, Urea in the cstraction solvent in the first column had almost no jnfluence on the yields of ribosotnes ancl viruses (compare proceclurc S with procedure 7),

PURIFICATION ,eorbencs

01: VIRUSES al254

AND RIDOSOMIZS RY cc

243

nm

lo0

0.5 lo0

0.8 lo0 0.4

1.0 0.6

1.0

0.5

1.0 0.5

1.0 03

1.0 0.1

1.0 05

1.0 0.5

1100 1

t400 3

lBO0 n

2100 5

ZPOU 0

1700 7

3000 II

3300 0

ml rolvonl

lIlturnt number

Pig, 3. Fractionntions of tohncco nlounic virus and potirto virus S from “1Vhitc lhrlcy” tobacco plnnts by the second step of the two-atcp chromatograpliic pmxlurcs on ccllulo~tc? coluinns. For oxplmatims, wc Fig. 1, Frilctions with a. niultiplicntion sign contain highly infectious tohcco tobncco plants nncl frnctions with it mowic virus gi\*ing typical symptoms on ” Wliitc Uurlcy” plus sign contain highly infectious potato virus S giving tvpical syniptonis on “White 13urloy” tolw.2c0 plants. A tobacco mosaic virw content of 100% ‘is cquivdcnt to an nbsorbnncc/ml/g to an abaorbancc/ml/y frc~ll wcigllt of II .38. A potato virus S content of 100’;: is cquivalcnt frc4h vfciglit of I++.

J, H. VENEICAMI’,

1.0

0.5

264 “y

L... ~beorbanGe 9

et

100

‘? 1200

4.9 4,s

0.m ._. cldl _._. s -5 06040.004 1 1 0.1

j;OO I 0.01 _.~ 0 0.004

:;00

d.01 ~.. 5

absorbEtW@

1200 4.5 4.5 0.01 0.01 55 Q0040.004 1 1 0.1

;5500 d.0t 5 0,004

at

100

i1500

‘3

132

I

W. H. Of. MOSCH, I’. TABORSICY

254

ii00 d.OC 3 0.004

6.01

;;OO

ii00

:CJOO 3300

0.01

do1 1

601

2

ml offluont % M % M % %

glucoao Trls PEG Ma(OAcl, NW,OAc NeDDC

mtw. nbbarbancc min. absorbonce

I.1 3

nm

f:OO 601 2 0.004

1 .to

%4500 ii00 o:ot d.01 1 1 0.004

1.14

1.20

3000 4.5 0,Ol

1.29

3300

ml affluent J ,$,,,o % PEG M MalOAd, % NH,CVx % NoDDC max. abrorbance min. absorbance

Fig, _I, Fmctionirtions of potato virus S antI ribosonws from “U%itc 13urlcy” tobacco plants by the second step of the two-step chromntographic proccclurcs on cullulosc colunlna. For cxphnations, sco Fig. I. l\ virus content of Ioo”/~ is cquivalfxt to an nbsorbnnw/tnl/g frcsli weight of 1.36. A ribosotnc content of x00”/, is cquivnlcnt to nn nl~sorbnncc/nil/g Frcali weight of ‘~~07.

The presence of 0.5% polycthylenc glycol in this solvent decreased tllc final yields of viruses and ribosomes (compare procedure 9 with proccclure S). In the experiment shown in Fig. I, procedures 7 and 8, and in the cxperimcnts shown in Pigs. ;z and 3, procedure 7 resulted in the IGghest yields of viruses and ribosomes. The measured alxorlxuices of the effluents were averaged. Tnblc III gives the characteristics of the absorption spectra of ribosomes ancl viruses,

PURIPICATION

OP VIRUSES AND

RIBOSOBIES BY cc

245

Tobacco ribosomes had a smaller ratio of maximum and minimum absorbances than bean ribosomes. In the experiment in Fig. 4, the elution of J3otato virus X prior to that of ribosomes was compared with proceclure 7 in Pig. 1. In this case, potato virus X was eluted by two successive solvents. The total yield of this virus was lower tllan in Jxocedure 7. When magnesium ions were omitted from the solvent, the ribosomes were eluted from the column. Moreover, the first ribosome fraction contained some virus particles according to the low ratio of maximum and minimum absorban.ces.

DISCUSSION

Precipitation of plant viruses by a mixture of Jx~lycthylene glycol and sodium chloride was reported by HEBIZRT~", VENEKAMP~~ used this technique in order to purify J>otato virus S, The UV absorption sJ>ectrurn of the final preparation, howover, was not typical of this virus in sJ>ite of the biological activity. %EPPTSZAUEIZAND BRISIIAMAIAR~" J~uriliccl glycoprotcins by using polyctllylcnc glycol as a Jxccipitant. Por.soN cl nl. 21 seclimcntecl Jxoteins with polycthylenc glycol. In both instances the concentrations of this polymer were similar to those required for the purification of viruses. Therefore, the impurity of precipitated potato virus X could be explained as tile result of coprccipitation of the virus with other cell components. This was the reason why the present authors preferred cellulose columns to separate viruses and other nucleoproteins. The techiqucs clescribcd in this Jxq>cr arc considcrcd to bc cllromatographic Jxocedures. For the second column, ground chromatogmphic paper coulcl be usccl insteacl of cellulose powder. Large particles, such as chloroplasts or cvcn wholC cells suspcndecl in sucrose solutions, JJassecl through this column easily. Viruses in solvents that contained polycthylenc glycol and ammonium acetate (or sodium chloride or soclium acetate), however, coulcl not pass through this column hcausc of aclsorption by the cellulose. Cellulose powder was usccl in the seconcl columns to obtain a regular pattern in the clironiatogran~s, The use of sodium dietJ~ylclithiocarbarn~~tc rcsultecl in a higher yield of potato virus S and white clover mosaic virus 1)ecause of the reversibility of the adsorption of reducccl phenols on the virus particles, Irreversible adsorption of osiclizecl phenols hindcrcd to some extent tile liberation of virus particles from the column. Sodium diethylditl~iocarbamate had, on the contrary, no cffcct on the yield of tobacco mosaic virus. In all instances, omission of magnesium ions resultecl in isolation of ribosomes, except when tobacco mosaic virus was Jxesent. Synthesis of this virus seemed to reduce appreciably the ribosome content in a plant. The omission of magnesium ions also inclucecl tile elution of potato virus S and white clover mosaic virus wit11 solvents containing a higher concentration of J)olyethylcne glycol. Therefore these viruses were eluted earlier. The presence of microsornal material in tllc column slowccl clown the liberation of potato virus X and white clover mosaic virus. The experiment shown in Fig. 4 supports this observation, In the proceclures described in this J’apcr, aggregation of the virus particles

246

J, H. VISNl1KAhIP,

W.

H. &I. IIIOSCH,

V. TABOIISKY

did not intcrfcrc in tlic liberation from tlw columns, hcausc urea had no cffcct on tlic clution, The rihsonies hm tobacco have tlic sonic ratio of maximum and mininiunl absorbanccs as tliose of fig fruits as found by iVhnw ANI) RohIAN122, Bean ribosomes of have a siniihr ratio to tlint of clover ribosonws 2:1,The ratios of these nbsorbanccs tobacco and bean ribosonws may indicate a cliffcrence in purity or in composition. Tile similarity of the ratios of the absorbawes at ZSO and zgS nrn of tllc ribosomes from tobacco and from lxxw plants inclicatcs no difference in tllc adsorption of phenolic conilx~uncls. All tlic proccclurcs yiclclecl fairly pure viruses. Cnrrcsponcling fractions isolntcd from healthy plant lioiiiogcnatcs were free from UV-nbsorl)ing nislterinls. Electron n\icrowopic stuclics of tllc prcpwations failccl to sliow tlic prcscncc of impurities. Tile absorlltion spectra of tllc lxxqxuxtions were typical of purified viruses. All tlie purifiecl viruses induced typical symptoms when inoculated into susceptible llosts. Iiil~wxncs from Iicalthy plants coulcl also lx isolated after tlie omission of magncsiunl ions. The electron microgrnplls and tile absorption spectra of tllc ribosomcs isolatecl in tllc cxlxAnlcnts clcscribccl wcrc typical of purified nuchproteins. From tlic alxwc! considerations it is eviclent that in procedure number 7, tlw 0lAimuni conditions to give tile liiglwst yiclcls of virus particles and ribosonics were realized.

Tlic authors arc inclcbtccl to Dr. G. IV. hllr.trsI~, Husley College of Environmcntnl Stticlics, Rellinglinin, Wasl1ington, U.S.A., for llis corrections to tllc test. They acknowledge tile teclmical assistance of Mrs. A. A. GIEHEM-SWIER and tllc suggestions of Miss Dm. J. RI. I